Magnetic nanoparticles, magnetic detector arrays, and methods for their use in detecting biological molecules

ABSTRACT

Magnetic nanoparticles and methods for their use in detecting biological molecules are disclosed. The magnetic nanoparticles can be attached to nucleic acid molecules, which are then captured by a complementary sequence attached to a detector, such as a spin valve detector or a magnetic tunnel junction detector. The detection of the bound magnetic nanoparticle can be achieved with high specificity and sensitivity.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional Application of U.S. patentapplication Ser. No. 10/829,505, filed Apr. 22, 2004, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/519,378,filed Nov. 12, 2003, the contents of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The government may own rights in the present invention pursuant to grantnumber N00014-02-1-0807 from the U.S. Defense Advanced Research ProjectsAgency (DARPA).

FIELD OF THE INVENTION

The invention relates to magnetic nanoparticles, magnetic nanoparticledetectors, and methods of detecting biological materials, whethernatural or synthetic, and whether modified or unmodified. The inventionalso relates to the magnetic nanoparticle materials for use in detectingbiological materials, and methods for making those materials. Finally,the invention relates to magnetic particle detectors and relatedapparatus, as well as methods of using such apparatus for the detectionof biological materials.

DESCRIPTION OF RELATED ART

The development of high sensitivity, quantitative DNA fragment detectionand identification systems has been of growing importance in the fieldsof functional genomics, forensics, bio-defense, anti-bioterrorism, andother biotechnology applications.

Ideally, detection systems should be sensitive, rapid, portable,inexpensive, and reusable. Additionally, it is preferable that thesystems do not require DNA amplification processes such as thepolymerase chain reaction (PCR). More specifically, the system shouldpreferably have the following characteristics: (1) one DNA fragment pertag, (2) each tag is individually detectable, (3) an effectivelyinfinite number of detectors, and (4) known efficiency of the attachmentprocesses involved. Currently, no system is commercially available thatsatisfies all of these requirements.

Numerous of the current microarray systems utilizing fluorescentlabeling (tagging) are inherently of low sensitivity because theyrequire approximately 10⁴ molecules to achieve a useful signal to noiseratio (thereby violating the ability to have each tag be individuallydetectable) and are only marginally quantitative because of the opticalsystems involved, crosstalk, and bleaching (M. Schena, R. W. Davis,Microarray Biochip Technology, Eaton Publishing, pp. 1-18 (2000)).Further, the optical detection systems are usually used in conjunctionwith the polymerase chain reaction (PCR).

However, several groups have recently taken a new approach to detectingtarget molecules. In U.S. Pat. No. 5,981,297 to Baselt (issued Nov. 9,1999), a group at the Naval Research laboratory offered both anapparatus and methods for detecting target molecules using amagnetoresistive or magnetostrictive magnetic field sensor havingbinding molecules attached which are reported to selectively bind targetmolecular species, which in turn are attached to paramagnetic polymerbeads.

In a related published article by D. R. Baselt, et al., entitled “ABiosensor Based on Magnetoresistance Technology”, (Biosensors andBioelectronics, Vol. 13, no. 7-8: 731-739 (1998)), a magnetic detectionsystem which they called BARC (Bead Array Counter) is offered. Accordingto the article, the BARC measures the forces that bind molecules such asDNA together, and use these interactions to hold magnetic microbeads toa solid substrate. Microfabricated magnetoresistive transducers on thesubstrate are reported to indicate whether the beads are removed whenpulled by magnetic forces, and can be adapted to chips for use inmulti-analyte detection capabilities.

M. M. Miller, et al., (“A DNA Array Sensor Utilizing Magnetic Microbeadsand Magnetoelectronic Detection”, Journal of Magnetism and MagneticMaterials, 225: 138-144 (2001)) offers a multi-analyte biosensor thatuses magnetic microbeads as labels to detect DNA hybridization on amicro-fabricated chip. The beads are detected using giantmagnetoresistance magnetoelectronic sensors that are embedded in thechip itself, allowing for the simultaneous detection of eight differentanalytes.

In U.S. patent application Ser. No. 2002/0060565 (published May 23,2002), Tondra suggests a ferromagnetic, thin-film based magnetic fielddetection system useful in detecting the presence of selected molecularspecies. According to the specification of this patent application, amagnetic field sensor is supported on a substrate that has a bindingmolecule layer positioned on a side of the substrate and is capable ofselectively binding to the selected molecular species.

Finally, a group in Portugal has deployed spin valve sensors coupledwith coils at proximity (D. L. Graham, et al., J. Appl. Phys., 91: 7786(2002)). The magnetic tags used were about 2 μm in diameter for theparamagnetic polystyrene balls, and similarly sized for theferromagnetic particles. The larger tags were coupled to a much largerand not easily ascertainable number of DNA fragments, prejudicing thequantitative capabilities of the system. The dimensions of the tags andof the magnetic detector suggested in this paper limit the detectordensity to levels 10² to 10⁴ less than the approach disclosed herein.

Despite the advances achieved so far, there still remains a need fordetection systems which ideally meet all four desirable qualities listedabove.

SUMMARY OF THE INVENTION

A system of magnetic nanoparticles and detector arrays are described.The system is useful for the high-sensitivity detection of nucleic acidmolecules such as DNA. The nanoparticles can be high moment magneticnanoparticles that are superparamagnetic, or antiferromagneticnanoparticles which contain at least two layers ofantiferromagnetically-coupled high moment ferromagnets.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A shows a schematic of a DNA detector comprised of a DNA probe(binding molecule) and a spin valve or magnetic tunnel junction (MTJ)detector, in accordance with one aspect of the present invention.

FIG. 1B shows a magnetic nanoparticle tag attached to a DNA fragment tobe detected (target molecule).

FIG. 1C shows the configuration of the DNA detector and the magneticnanoparticle tag after the target DNA and binding DNA are hybridized.

FIG. 2A shows a spin valve detector with a magnetic tag above it,illustrating the pinned layer magnetization along the y-direction. Thefree layer magnetization has an easy-axis along x and hard-axis along y.

FIG. 2B shows two modes of interrogation method of spin valve detector,the AC tickling field H_(t) is either parallel (in-plane mode) or normalto (vertical mode) the spin valve plane (x-y plane). H_(b) is the DCbias field.

FIG. 2C shows spin valve resistance change ΔR due to a single Conanoparticle as a function of the phase of the tickling field H_(t) inthe vertical detection mode.

FIG. 3A illustrates a magnified view of a 16-nm Fe₃O₄ nanoparticlemonolayer on a 0.3-μm spin valve (SV) sensor.

FIG. 3B illustrates a graph of the voltage signals of a 0.3-μm spinvalve (SV) sensor with a Fe₃O₄ nanoparticle monolayer and a sensorwithout nanoparticles; the line is the modeling result.

FIG. 4 shows the magnetoresistance (MR) and ΔR values for a spin valvesensor submerged in a blocking solution. As shown, the spin valvemaintains its MR ratio and ΔR after 24 hours of exposure to the blockingsolution.

FIG. 5 shows a schematic representation of a syntheticantiferromagnetically coupled magnetic nanoparticle with an under layer(Underlayer) and a gold cap, Au. The layers with arrows areferromagnetic layers which are antiferromagnetically coupled in theremanence state. The number of the ferromagnetic layers can vary fromabout 2 to about 6 depending upon the application. The gold cap is forbio-conjugation, while the under layer is for proper film growth as wellas biochemistry applications.

FIG. 6 illustrates an additive fabrication method of syntheticferromagnetic nanoparticles.

FIG. 7A shows microfluidic channels directly integrated on a longdetector, with the DNA probes attached specifically to the detectorsurface. As shown in the figure, the detector is made slightly largerthan 20 μm, the width of the microfluidic channel, while the longdetector is suitable for relatively large quantities of DNA samples.

FIG. 7B shows microfluidic channels directly integrated on a shortdetector, with the DNA probes attached specifically to the detectorsurface. The detector is made shorter than the width of the microfluidicchannel, so as to be more suitable for use with relatively smallquantities of DNA samples.

DEFINITIONS

The following definitions are provided in order to aid those skilled inthe art in understanding the detailed description of the presentinvention.

“Binding molecule”, as used herein, refer to antibodies, strands ofpolynucleic acids (DNA or RNA), and molecular receptors capable ofselectively binding to or ‘recognizing’ potential target molecules suchas polynucleic acids, enzymes, proteins, peptides, antibodies, lipids,polymers, metal ions, and low molecular weight organic and inorganicspecies such as toxins, drugs (both prescription and illicit),explosives, and biohazards.

“Target molecule”, or “target species”, as used herein, refers to themolecule, molecular species, or organism whose presence, absence, orconcentration the assay in question actually determines. Targetmolecules included for use with the present invention include but arenot limited to viruses, bacteria, other biological organisms such asfungi, antibodies, proteins, peptides, polynucleic acids, lipids,polymers, pharmaceutical compounds, organic compounds, biohazardouscompounds, explosive compounds, and toxins, among others.

“Detector”, as used herein, refers to any number of magnetic detectionsystems including spin valve detectors (also referred to as spin valvefilm detectors), magnetic tunnel junction (MTJ) detectors, and MagArray™detectors, as well as MagArray™ variants of both spin valve detectorsand MTJ detectors.

DETAILED DESCRIPTION OF THE INVENTION

A detection system can typically involve an array of spin valves or MTJdetectors, oligonucleotide probes complementary to a target of interestattached to individual detectors in the array as shown in FIG. 1A, amacrofluidic or microfluidic sample delivery system, and magneticnanoparticles bound with target DNA fragments as shown in FIG. 1B.Tagged DNA fragments are delivered by fluidic channels to the detectorarray for selective hybridization as shown in FIG. 1C. Non-hybridizedDNA fragments are washed away, or are removed by a magnetic fieldgradient. The detector array is interrogated with a combination of DCbias field and AC tickling field, as shown in FIGS. 2A and 2B. Theapplied fields cause the magnetic nanoparticle tags to display netmagnetic moments, which in turn can be picked up by the spin valve orMTJ detectors. In the in-plane detection mode, the detector signal hasthe same frequency as the AC tickling field H_(t). In contrast, in thevertical mode, the detector signal is a second harmonic of the ACtickling field as shown in FIG. 2C. In either case, lock-in detectioncan be employed even if the signal to noise ratio is small. The presenceof a magnetic nanoparticle tag, signaling the presence of a target DNAfragment, can thus be detected. The detector voltage signal isproportional to the number of magnetic nanoparticles, and therefore thenumber of target DNA fragments.

Generally, a DNA fragment can be tagged with a magnetic nanoparticle.The tagged fragment can be selectively bound to a substrate using acomplementary nucleotide above a spin valve. The spin valve is then usedto detect the magnetic nanoparticle.

The methods and systems disclosed herein are much more sensitive thanthe previously reported optical detection systems and the other magneticdetection schemes of the known prior art. The inventive systems are moreefficient and sensitive than BARC, since they involve spin valve or MTJdetector designs. Additionally, nanometer-scale particles of highmagnetic moment are used as biomolecule tags instead of larger particleswith more dilute magnetic material. The systems are more sensitive thanfunctional MRI (fMRI) systems. Calculations indicate that the amount ofgadolinium required for detection by fMRI far exceeds the amount ofmagnetic nanotags that can be detected with the inventive systems.Finally, the inventive systems do not require the sophisticated coolingapparatuses required for detection by SQUID (Superconducting QuantumInterference Device) detector systems.

Aspects of the invention include magnetic nanoparticles, detectors,detection systems, and methods for their use. Various aspects of theinvention are discussed below. U.S. Trademark application Ser. No.78285336 was filed on Aug. 9, 2003 for the mark MAGARRAY (ApplicantSunrise Associates).

Nanoparticles

Nanoparticles useful in the practice of the present invention arepreferably magnetic (i.e., ferromagnetic) colloidal materials andparticles. The magnetic nanoparticles can be high moment magneticnanoparticles which are small in size so as to be superparamagnetic, orsynthetic antiferromagnetic nanoparticles which contain at least twolayers of antiferromagnetically-coupled high moment ferromagnets. Bothtypes of nanoparticles appear “nonmagnetic” in the absence of magneticfield, and do not normally agglomerate. In accordance with the presentinvention, magnetizable nanoparticles suitable for use comprise one ormore materials selected from the group consisting of paramagnetic,superparamagnetic, ferromagnetic, and ferrimagnetic materials, as wellas combinations thereof.

The magnetic nanoparticles preferably possess the following properties:(1) their remnant magnetization is as small as possible so that theypreferably will not agglomerate in solutions (either superparamagneticparticles or antiferromagnetic particles can satisfy this requirement);(2) the tags display high magnetic moments under a modest magnetic fieldof about 100 Oe so they can be readily detected; (3) the size of thetags preferably is comparable to the target biomolecules so that they donot interfere with the DNA hybridization process and other biologicalprocesses; (4) the tags preferably are uniform and chemically stable ina biological environment; and/or (5) the tags preferably arebiocompatible, i.e., water soluble and functionalized so that they arereadily attached to DNA fragments or other biomolecules.

The nanoparticles are preferably high moment magnetic nanoparticles suchas Co, Fe or CoFe nanocrystals which are superparamagnetic at roomtemperature. They can be fabricated by chemical routes such as saltreduction or compound decomposition in appropriate solutions. Examplesof such magnetic nanoparticles have been published in the literature (S.Sun, and C. B. Murray, J Appl. Phys., 85: 4325 (1999); C. B. Murray, etal., MRS Bulletin, 26: 985 (2001)). These particles can be synthesizedwith controlled size (e.g., 5-12 nm), are monodisperse, and arestabilized with oleic acid. In accordance with the present invention, itis also possible to fabricate high magnetic moment nanoparticles in ananocluster deposition system (D. J. Sellmyer, et al., Chap. 7, Handbookof Thin Film Materials, edited by H. S. Nalwa, Academic Press (2002)).These particles have been developed for applications in bioconjugation.Magnetic nanoparticles and nanopowders suitable for use with the presentinvention include but are not limited to Co, Co alloys, ferrites, Cobaltnitride, Cobalt oxide, Co—Pd, Co—Pt, Iron, Iron alloys, Fe—Au, Fe—Cr,Fe—N, Fe₃O₄, Fe—Pd, Fe—Pt, Fe—Zr—Nb—B, Mn—N, Nd—Fe—B, Nd—Fe—B—Nb—Cu, Ni,and Ni alloys. Alternatively and equally acceptable, a thin layer ofgold can be plated onto a magnetic core, or a poly-L-lysine coated glasssurface can be attached to a magnetic core. Suitable nanoparticles arecommercially available from, e.g., Nanoprobes, Inc. (Northbrook, Ill.),and Reade Advanced Materials (Providence, R.I.).

Magnetic nanoparticle tags can be fabricated by physical methods insteadof chemical routes, and are suitable for labeling the targetbiomolecules to be detected. The tags comprise at least two thinferromagnetic layers, preferably Fe_(x)Co_(1-x), wherein x is 0.5 to0.7, or Fe_(x)Co_(1-x) based alloys. Fe_(x)Co_(1-x) has the highestsaturation magnetization (about 24.5 kGauss) among the knownferromagnetic materials (R. M. Bozorth, Ferromagnetism, D. Van NostrandCompany (1951)). These ferromagnetic layers are separated by nonmagneticspacer layers such as Ru, Cr, Au, etc., or their alloys. The spacerlayers are appropriately engineered to make the ferromagnetic layerscoupled antiferromagnetically so that the net remnant magnetization ofthe resulting particles are zero or near zero. The antiferromagneticcoupling can be achieved via RKKY exchange interaction (S. S. P. Parkin,et al., Phys. Rev. Lett., 64 (19): 2304 (1990)) and magnetostaticinteraction (J. C. Slonczewski, et al., IEEE Trans. Magn., 24 (3): 2045(1988)) as practiced in the magnetic data storage industry. However, theantiferromagnetic coupling strength preferably is moderate so that theparticles can be saturated (i.e., magnetization of all layers becomeparallel) by an external magnetic field of about 100 Oe. This can beachieved by adjusting layer thicknesses and by alloying the spacerlayer.

To facilitate the bio-conjugation of the nanoparticle, a gold cap can beadded at the top of the antiferromagnetic stack so that the nanoparticlecan be conjugated to biomolecules via the gold-thiol linkage.Furthermore, appropriate surfactants can also be readily imparted to thenanoparticles, rendering them water-soluble. The edge of thenanoparticles can be passivated for chemical stability with Au or otherthin inert layers.

Many physical methods can be conceived by those familiar with the art tofabricate the nanoparticles described above. A film stack can be made ofnanometer-scale ferromagnetic and spacer layers, so they need to bedeposited on ultrasmooth substrates (or release layer). The mask layercan be formed by imprinting, etching, self assembly, etc. Subsequentlythe mask layer and unwanted film stack are removed and cleaned offthoroughly. Then, the release layer is removed, lifting offnanoparticles which are the negative image of the mask layer. Theseparticles are eventually imparted with surfactants and biomolecules. Theultrasmooth substrate can be reused after thorough cleaning and chemicalmechanical polishing (CMP).

Alternatively, the nanoparticles can be fabricated with a subtractivefabrication method. In this case, the film stack is directly depositedon the release layer followed by a mask layer. The film stack is etchedthrough the mask layer, and eventually released from the substrate.These nanoparticles result from a positive image of the mask layer asopposed to the case in the additive fabrication method.

The size of the magnetic nanoparticles suitable for use with the presentinvention is preferably comparable to the size of the target biomoleculeto be worked with, such that the nanoparticles do not interfere withbiological processes such as DNA hybridization. Consequently, the sizeof the magnetic nanoparticles is preferably from about 5 nm to about 250nm (mean diameter), more preferably from about 5 nm to about 150 nm, andmost preferably from about 5 nm to about 20 nm. For example, magneticnanoparticles having a mean diameter of 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 70 nm, 80nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, and 150 nm, as wellas nanoparticles having mean diameters in ranges between any two ofthese values, are suitable for use with the present invention. Further,in addition to the more common spherical shape of magneticnanoparticles, nanoparticles suitable for use with the present inventioncan be disks, rods, coils, or fibers.

Synthetic antiferromagnetic nanoparticles for use in this applicationmay be considerably larger than ordinary ferromagnetic particles. Thisis because, to prevent clumping, the nanoparticle must have no netmagnetic moment (or a very small magnetic moment) in zero applied field.Antiferromagnetic particles may have zero magnetic moment in zero fieldat all sizes, but for a ferromagnetic particle its size must be belowthe “superparamagnetic limit”, which is typically 20 nm or less, usuallyless. To demonstrate the advantage of the synthetic antiferromagneticparticle we have made a calculation of the voltage produced in a spinvalve detector for a synthetic antiferromagnetic particle 30 nm indiameter and 30 nm in height and compared it to the voltages produced by16 nm diameter Fe₃O₄ and 11 nm diameter Co nanoparticles. We assume that75% of the synthetic antiferromagnetic particle is ferromagnetic FeCoand that the spin valve detector is the same in all cases. Table 1 givesthe results of these calculations and the caption of Table 1 gives thespin valve dimensions and operating conditions. Note that the spin valvesignal from the synthetic antiferromagnet is nearly two orders ofmagnitude greater than for the ferromagnetic particles.

TABLE 1 Spin valve signal voltage (peak-to-peak amplitude) versusmagnetic tag. Only the data for the vertical detection mode (FIG. 2B) islisted here. The voltage is due to a single nanoparticle with its center20 nm away from the midplane of the spin valve free layer. The sensorsize is 3 μm × 0.2 μm with an active length of 1 μm. The sense currentdensity is 10⁸ A/cm². The effect of the stray field from the spin valvesensor is included in the calculation. The synthetic FeCo is assumed tosaturate at 30 Oe and the particle physically rotates with the appliedfield. Note that the room temperature magnetic moments of thesuperparamagnetic nanoparticles are reduced as described by the Langevinfunction, but that of the synthetic antiferromagnetically couplednanoparticles is changed much less by superparamagnetism. ParticleSynthetic FeCo Fe₃O₄ Co Saturation magnetization 1950 emu/cc 480 emu/cc1400 emu/cc Size 30 nm diameter, 16 nm 11 nm diameter 30 nm heightdiameter Net saturation moment 31 femtoemu @ 1.0 femtoemu @ 1.0 femtoemu@ 75 vol % 100 vol % 100 vol % magnetic Vertical mode SV 193 μV 0.9 μV0.8 μV detector signal (bias field 100 Oe, tickling field 141 Oe)

Note that the signal levels listed in Table 1 are for spin valvedetectors. If replaced with an MTJ detector with a junction area of 0.2μm by 0.2 μm and resistance-area product of 1 kOhm-μm², operating with amagnetoresistance (MR) of 25% at a bias voltage of 250 mV, and H_(b)=35Oe, H_(t)=100 Oe rms, the voltage signal from a single synthetic FeConanoparticle could reach greater than 1 mV. This signal level makes itdetectable without a lock-in amplifier, greatly speeding up the entireMagArray™ detector readout process.

In addition to their advantageous signal level, the syntheticantiferromagnetically coupled nanoparticles can be saturated indifferent applied fields. This feature can be exploited for multiplexmagnetic separation of cells. For the MagArray™ detector, differentkinds of synthetic antiferromagnetically coupled nanoparticles with aseries of saturation threshold fields can be used to label thebiomolecules from different biological processes, thus achievingmultiplex biological analysis such as “multi-color” gene expressionanalysis. For example, consider a “two-color” gene expression schemewith two types of magnetic particles, one saturating in 100 Oe and theother in 125 Oe. We can then interrogate the MagArray™ detector with atwo-test sequence. The first test saturates the first type of magneticparticles and gives a voltage signal V₁, then the second test saturatesboth types of magnetic particles and gives a voltage signal V₂. Bothtypes of particles contribute to the signals measured in the tests. Ifthe numbers of the two types of particles at a given site are N₁ and N₂,respectively, then the tested voltage signals should be:V ₁=α₁₁ ×N ₁+α₁₂ ×N ₂,V ₂=α₂₁ ×N ₁+α₂₂ ×N ₂,

where α_(ij) (ij=11, 12, 21, or 22) are calibration constants. Bysolving the above equations, we can quantify both types of particles andthe two types of genes (or other biomolecules) tagged to them.

The synthetic nanoparticles described above can be produced in largequantities using a large wafer and standard vacuum thin film depositionprocesses. For example, with a 6-inch round wafer, we can produce 30-nmdiameter nanoparticles at a rate of roughly 5×10¹² particles per run,assuming each particle occupies a square of 60 nm by 60 nm on the wafer.

High Sensitivity Spin Valve Detectors

A spin valve detector is a metallic multilayer thin-film structure oftwo ferromagnetic layers spaced by a non-magnetic layer such as copper.One ferromagnetic layer, called the pinned layer, has its magnetizationpinned to a certain direction, while the magnetization of the otherferromagnetic layer, called the free layer, can rotate freely under anapplied magnetic field. The electrical resistance of a spin valvedepends on the relative orientation of magnetization of the free layerto that of the pinned layer. When the two magnetizations are parallel,the resistance is the lowest; when antiparallel, the resistance is thehighest. The relative change of resistance is called themagnetoresistance (MR) ratio. The MR ratio of a spin valve can reachmore than 10% in a small magnetic field, e.g., 100 Oe. Therefore, a spinvalve is a good sense element for the detection of a small magneticparticle that is attached to a DNA fragment as a label and immobilizedonto the sensor surface. Since the particle is magnetic (under a DC biasfield), it generates a magnetic field. The magnetic field may thenaffect the orientation of the free layer magnetization, causing a changein the electrical resistance of the spin valve.

The operation of a spin valve detector (FIGS. 2A and 2B) is described asfollows: 1) The magnetic nanoparticle under a DC bias field (H_(b))generates a magnetic field around it. 2) The magnetic field will affectthe resistance of a spin valve closely underneath it. 3) Application ofan AC tickling field (H_(t)) will force the moment of particle tooscillate, resulting in an oscillating MR signal from spin valve. Notethat in the in-plane mode the spin valve detector signal due to themagnetic nanoparticle has the same frequency f as the AC tickling fieldH_(t), while in the vertical mode the signal has twice the frequency ofH_(t). 4) A lock-in amplifier is used to pick up such an oscillatingsignal with a high signal-to-noise ratio.

Spin valves have a magnetoresistive (MR) ratio of typically 5-12% andare used in hard disk drives to detect a magnetic bit made of only a fewhundred closely packed Co alloy nanoparticles (size is about 10 nm) witha signal to noise ratio (SNR) of about 20 dB and a broad bandwidth ofabout 500 MHz. Therefore, it is theoretically feasible to detect asingle Co nanoparticle of about 10 nm size in a narrower bandwidth orwith lock-in detection. By narrowing the noise bandwidth, sufficient SNRis achieved even for single nanoparticle detection.

As a proof of concept, a prototype spin valve detector and detectorarrays were prepared with a sensor width of about 1 μm or less (alongthe direction normal to the sense current through the spin valve and tospin valve thickness). It has been demonstrated that, after applying adiluted drop of 11-nm diameter Co nanoparticle dispersion on suchdetectors, we can obtain a signal amplitude of greater than 1 mV(peak-to-peak) from a 1 μm wide spin valve detector (Li, G., et al.,Journal of Applied Physics, Vol. 93, no 10 (2003), p. 7557). Thesensitivity of the spin valve detector depends not only on themagnetization and volume of magnetic tags and their distance from thefree layer of the spin valve, but also on the geometry and bias field ofthe spin valve itself. We have found that narrower spin valves generallylead to a higher sensitivity. Consequently, spin valve detectors anddetector arrays suitable for use with the present invention have sensorwidths from about 0.01 μm to about 1 μm along the direction normal tothe sense current and to spin valve thickness.

We have performed micromagnetic and analytical simulations of variousspin valve designs extensively and summarize the signal (peak-to-peakamplitude prior to any preamplifier) due to a single Co nanoparticleversus spin valve free layer width in Table 2. Both the in-plane andvertical modes of operation (FIG. 2B) are listed. The distance from theparticle edge to the midplane of the free layer is assumed to be 6 nm.The free layer strip is 2 nm thick and 3 μm long, but its active length(not covered by leads) is 1 μm. The sense current density is taken to be10⁸ A/cm², which is below the electromigration limit of the spin valvedetector. The total detector thickness is about 34 nm. The magneticmoment of superparamagnetic Co nanoparticles have been calculated withthe Langevin function.

We should have a sufficient signal level to detect a single11-nm-diameter Co nanoparticle if the spin valve is made to be 0.2 μmwide and operated in the in-plane mode. Additionally, we can increasemagnetic signal strength further by using FeCo-based magnetic nanotags,since the signal voltage is proportional to the magnetic moment in thetag.

TABLE 2 Spin valve signal voltage (peak-to-peak amplitude) versus freelayer width. The voltage is due to a single Co nanoparticle with adiameter of 11 nm and its edge is 6 nm away from the midplane of thespin valve free layer. Both in-plane and vertical mode are listed, alongwith the relevant bias field and tickling field amplitudes. Free layerwidth 1 μm 0.2 μm 0.2 μm Sense current 10 mA 2 mA 2 mA In-plane (biasfield, (100 Oe, (100 Oe, (100 Oe, mode tickling field) 50 Oe) 50 Oe) 141Oe) signal voltage 0.32 μV 2.1 μV 4.9 μV Vertical (bias field, (50 Oe,(50 Oe, (100 Oe, mode tickling field) 50 Oe) 50 Oe) 141 Oe) signalvoltage 0.08 μV 0.2 μV 0.8 μV

Electromagnetic Interference (EMI) Signal Rejection

Spin valve detection is typically performed with the in-plane mode (Li,et al., J. Appl. Phys. Vol. 93 (10): 7557 (2003)). The vertical mode,even though giving a slightly smaller signal amplitude, is extremelyadvantageous when the electromagnetic interference (EMI) signal due tothe AC tickling field in the detection system is significant. The EMIsignal tends to center at the frequency f of the AC tickling field, soit can be eliminated or greatly reduced if we perform lock-in detectionat the frequency 2f. Furthermore, we can adopt a 2-bridge circuit toeliminate any remaining EMI.

Ultrathin Passivation of Detectors

The signal from the spin valve detector due to the magnetic tag dependson the distance between the magnetic tags and the free layer of the spinvalve, in addition to the geometry and bias field of the spin valveitself. The detector voltage signal from a single Co particle decreaseswith increasing distance from the center of the particle to the midplaneof the spin valve free layer.

As the sensing magnetic field from a magnetic particle dropsmonotonically with the distance between the sensor and the particle, itis preferred to make the free layer in the spin valves on top of thepinned layer. Furthermore, it is of utmost importance to minimize thedistance between the magnetic particle and the top surface of the freelayer, including the thickness of the passivation layer protecting thespin valves. However, during operation of the detector array, a solutionof DNA will be flowed over the sensor surface to allow for hybridizationof corresponding DNA fragments. Therefore, corrosion of the sensorsurface is of major concern. Any degradation of the detector surfacecould sacrifice sensitivity by reducing the signal from hybridizationevents or by destroying the detectors altogether.

The magnetic detection schemes in the prior art have recognized thispotentially catastrophic problem and consequently have added relativelythick passivation layers to their detector surfaces. If a conventionalpassivation layer is used, there would be a distance of greater than1000 nm between center of the magnetic particle and the detectorsurface, greatly limiting the detector sensitivity. A trade-off occursbetween retaining high sensitivity while sufficiently guarding againstdegradation. The MagArray™ detector design combines an ultrathin (lessthan 10 nm) layer of passivation and very small magnetic nanoparticletags (diameter of about 20 nm or smaller), thus achieving aparticle-center-to-detector distance of less than about 30 nm (includingthe intervening DNA fragment length of about 10 nm), which is closeenough to provide the necessary sensitivity for single-tag detection. Inaccordance with the present disclosure, the ultrathin layers ofpassivation (such as Ta or Au) suitable for use with detectors such asthe MagArray™ detector typically can have a thickness from about 1 nm toabout 10 mm, allowing for achievement of particle-center-to-detectordistances from about 6 nm to about 30 nm.

High Sensitivity MTJ Detectors

A MTJ detector is constructed similarly to a spin valve detector exceptthat the non-magnetic spacer is replaced with a thin insulating tunnelbarrier such as alumina and that the sense current flows perpendicularto the film plane. Electron tunneling between two ferromagneticelectrodes is controlled by the relative magnetization of the twoferromagnetic electrodes, i.e., tunneling current is high when they areparallel and low when antiparallel. A typical MTJ detector is composedof a bottom electrode, magnetic multilayers including a tunnel barrier,and a top electrode. MTJ detectors have magnetoresistance ratios as highas 50% and inherently large device resistances, yielding higher outputvoltage signals.

Conventional MTJ devices employ relatively thick (greater than 0.2 μm)top electrodes (Parkin, S. S. S. P., et al., J. Appl. Phys. 85: 5828(1999)) that will greatly reduce the detected signal from a singlemagnetic nanoparticle, thus they are not suitable for the MagArray™detector. To overcome this problem, we devised a double-layer topelectrode. The first layer can be a thin gold layer (about 10 nm orless). Gold is desirable due to its ease for binding DNA probes. Thesecond layer can be aluminum, copper or other conductive metals which donot bind with DNA probes, including palladium, palladium alloys,palladium oxides, platinum, platinum alloys, platinum oxides, ruthenium,ruthenium alloys, ruthenium oxides, silver, silver alloys, silveroxides, tin, tin alloys, tin oxides, titanium, titanium alloys, titaniumoxides, and combinations thereof. An aperture in the second layer,slightly smaller in size than the MTJ, is created either by a lift-offprocess or by etching a uniform second layer. This design allows us tokeep the distance between the nanoparticle tag and the top surface ofthe free magnetic layer very small, from about 6 nm to about 30 nm.Furthermore, this could circumvent current crowding (van de Veerdonk, R.J. M., et al., Appl. Phys. Lett., 71: 2839 (1997)) within the topelectrode which would likely occur if only a very thin gold electrodewere used.

Except that the sense current flows perpendicular to the film plane, theMTJ detector can operate similarly to the spin valve detector, eitherwith in-plane mode or vertical mode. The disclosure on EMI rejection andultrathin passivation also applies to MTJ detectors, but to theadvantage of MTJ detectors, the first top electrode of thin gold on MTJalso serves the triple purposes of electrical conduction, ultrathinpassivation as well as specific DNA probe attachment.

At the same detector width and particle-detector distance, MTJ detectorsgive appreciably larger signals than spin valve detectors. For example,for an MTJ detector with a junction area of 0.2 μm by 0.2 μm andresistance-area product of 1 kOhm-μm², operating with a MR of 25% at abias voltage of 250 mV, and H_(b)=35 Oe, H_(t)=100 Oe rms, the voltagesignal from a single 11 nm diameter Co nanoparticle whose center is 35nm away from the free layer midplane is about 20 μV, roughly an order ofmagnitude larger than those listed in Table 1 for similar-sized spinvalve detectors. This is a great advantage for MTJ detectors over spinvalve detectors. In accordance with the present invention, MTJ detectorssuitable for use in practicing the invention can have junction areasfrom about 0.01 μm² to about 10 μm², and resistance area products fromabout 0.1 kOhm-μm² to about 100 kOhm-μm².

DNA Quantification and Dynamic Range

Single-tag detection has been previously demonstrated bothexperimentally (Li, G., et al., Journal of Applied Physics, Vol. 93, no10 (2003), p. 755.7) and theoretically (Li, G., et al., IEEE Trans. MAG,Vol. 39, no. 5 (2003), p. 3313) in prototype MagArray™ detectors. Inreal applications, however, multiple particles may be on a detector, andtheir locations are likely not at the center of the detector surface. Wefound that the voltage signal from a single particle strongly depends onits lateral location on the detector surface, more so in the hard-axisdirection (y-axis in FIG. 2A) of the free layer than in the easy-axis(x-axis in FIG. 2A). The calculated time-domain voltage signal under asinusoidal tickling field versus the y-axis of an 11 nm diameter Conanoparticle whose center is 25 nm away (z-axis in FIG. 2A) from the topsurface of the free layer can be measured. The 2f signal gets distortednear the sensor edge and drops rapidly at the edge. Note that theoverall detector signal is larger at the detector edge, but it consistsof mostly 1f components which are out of phase at the two edges.

In order to count the magnetic nanoparticles quantitatively based on theamplitude of the voltage signal, we want each particle to generate thesame signal regardless of its location. A spin valve detector may notallow us to quantify the number of particles accurately. The MagArray™detector adopts a detection window which removes the nonuniformity nearthe edge. Note that the two-layer top electrode designs for MTJ canremove the edge uniformity as long as we make the Au window sufficientlysmaller than the active junction. Depending on the detector geometry andthe tolerance to signal variations, coverage of about 50% of activedetector area may be desired.

The detection window described above allows the MagArray™ detector tocount multiple nanoparticles (NP) very well. Based on the assumptions ofequivalent averaged magnetic field of NPs and coherent magnetizationrotation of the free layer, we have calculated the detector signals ofmultiple magnetic NPs uniformly or randomly distributed over arectangular area somewhat smaller than the active detector area. Forexample, for a 4×0.3 μm² spin valve (SV) detector, the normalizedsignals of uniform NP arrays versus the actual NP numbers for differentarray aspect ratios can be determined. At low particle numbers thesignal is fairly linear, and only at high particle numbers does thesignal linearity degrade. A higher aspect ratio gives better signallinearity because more NPs are away from the sensor edges. The meanvalues and standard deviations of the normalized signals for randomlydistributed NPs can be determined, which also indicates good signallinearity. We have done experiments on a monolayer of 16-nm Fe₃O₄ NPscoated on 0.3-μm wide SV sensors to verify the model.

These results indicate that the detector can not only detect 1-10 NPsbut also count hundreds of NPs with a resolution of a few NPs, farexceeding the detection limit of state of the art optical microarrays.Furthermore, we could multiplex a set of detectors with various sensorwidths and lengths such that the smaller detectors can sense lowconcentration biomolecules while the larger ones can sense and counthigh concentration biomolecules.

Nucleic Acid Probes Attached to Detector

An additional embodiment of the invention is directed towards nucleicacid probes (e.g. DNA or RNA) attached to a detector.

An oligonucleotide probe (binding molecule) complementary to the DNAfragment of interest (target molecule) is attached to the surfacedirectly above the detector via a 5′ linkage. To this end, the surfaceof the detector may be coated with a thin layer of glass or gold, andprobe DNA may be attached. The attachment of probe DNA to glass surfacesvia poly-L-lysine or to gold surfaces via a thiol linkage is widelypracticed in biochemistry. Alternatively, a monolayer of self-assembledprobe DNA can be prepared directly on the surface of the detector withno additional fabricated layer between the detector and the probe DNA.

The 5′ end of the target DNA is labeled with a magnetic nanotag. Duringthe hybridization step, the magnetically labeled target DNA attachesspecifically to the immobilized probe DNA on the surface of thedetector. It is highly preferred that the distance between magnetic tagand the free layer in the detector be kept to a minimum, since strengthof the magnetic signal will be compromised if the distance between thedetector and the signal is too great. One scheme is to apply an externalmagnetic field gradient during the hybridization step to concentratetarget DNA and to pull the magnetic tag closer to the detector surfaceafter the hybridization step. The external magnetic field may also beused to remove non-hybridized tagged DNA fragments.

Array Architecture

We have designed an architecture for the MagArray™ detector that issuitable for quantitative detection of DNA fragments. To utilize thesubstrate area efficiently, we have employed a scheme that appearssimilar to magnetic random access memory (MRAM), but in fact theMagArray™ detector is distinctively different from MRAM in that theMagArray™ detector does not require conduction lines for write currentsand that the signal levels in the MagArray™ detector are much smaller.The cells in the MagArray™ detector need to share preamplifiers and alock-in amplifier (or a narrowband pass filter) so that signals as smallas about 1 μV can be reliably detected. Each detector is connected tothe drain of a switching transistor and each cell framed by one rowconduction line (word line) and one column conduction line. We can readan individual detector by turning on the corresponding transistor andpassing a sense current through the detector. The voltage change in thecolumn lines are detected by the preamplifier and the lock-in amplifier.

A typical block with 1024 cells in the MagArray™ detector has beendesigned. Each block consists of a row decoder, a column decoder, apreamplifier, current sources, and an array of cells. At least onecolumn of cells are covered with thick polymers, rendering theminsensitive to magnetic nanoparticles, and thus can be used as referencedetectors in a bridge circuit or a subtraction circuit. We estimate thatthe MagArray™ detector can have a density of as high as about 10⁶cells/cm² (one detector per cell).

The DNA probes can be immobilized on the MagArray™ detector chips byconventional spotting or ink-jet printing. Each circular feature of DNAprobes (or binding molecules) spans over a multiplicity of cells, butthe probes bind only to the active detector area of each sensor withinthe feature. There is at least one feature per DNA probe to capture acorresponding DNA target. Unspecifically bound probes are then washedaway without cross contamination. After hybridization with magneticallytagged DNA target samples, each detector within the same DNA featurewill be interrogated individually, the resulting average signal is usedto identify and quantify the DNA target captured by the DNA probes at agiven site. In spite of the high density of chemically active sensorsurfaces, they still occupy only a fraction of the cell area because adetector is much smaller than a cell.

Alternatively, to improve the chemical sensitivity of the MagArray™detector, we have integrated microfluidic circuits directly onto thedetector array so that DNA probes and target samples are only attachedor directed at the detector surfaces which are sensitive to magnetictags. Here we may achieve one detector (instead of one feature) per DNAprobe, maximizing the number of different DNA probes which can beaccommodated on a chip. Several methods exist to attach DNA probesspecifically to surfaces such as silicon dioxide or gold which areselectively deposited on active detector surfaces only. This approachwill greatly boost the overall chemical sensitivity, allowing us todetect minute amount of biomolecules, e.g., 1-10 target DNA fragments,to minimize the amount of DNA probes spotted on the MagArray™ detector,and to speed up the detection process.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 Detection of Magnetite Nanoparticles

A series of experiments was carried out to demonstrate that in additionto Co, Fe, and their alloys, ferrite such as magnetite and Mn-ferritecan also serve as biological tags, in accordance with the presentinvention. A monolayer of 16-nm Fe₃O₄ nanoparticles (NP) on 0.3-μm widespin valve sensors was coated using polyethylenimine (PEI)-mediatedself-assembly method (see, S. Sun, et al., J. Am. Chem. Soc., 124, 2884(2002)), as shown in FIG. 3A. It was found that the voltage signal froma spin valve covered magnetite nanoparticles are nearly proportional tothe bias voltage applied to the sensor as expected, shown in FIG. 3B,while the signal from a reference spin valve not covered with anymagnetite nanoparticles is nearly zero. The voltage signals weremeasured from a Wheatstone bridge circuit by a lock-in amplifier atdifferent bridge circuit biases. Furthermore, it is demonstrated thatthe measured signals could be well described by an analytical model,such as described by G. X. Li and S. X. Wang in IEEE Trans. Magn., 39(5), 3313-5, (2003).

The detection of hundreds of magnetite nanoparticles in a patternedmonolayer by a spin valve sensor was also demonstrated. It was found,using an experiment similar to that described above, that the maximumresistance change of the spin valve due to about 630 magnetite particlesself assembled at the top surface of the spin valve was about 1.3Ω. Inother words, the signal per particle was roughly 2 mΩ, which isequivalent to 2 μV of signal voltage if the sense current is 1 mA. Thedetection limit in this experiment was approximately 55 mΩ, suggestingthat the minimum detectable number of the magnetite nanoparticles isabout 30. However, it seems apparent that with a more sensitive sensor,such as magnetic tunnelling junctions and higher moment nanoparticlessuch as FeCo, an even lower detection limit of nanoparticles isreachable. Consequently, it is readily evident from this example that itis realistic to detect from tens of magnetic nanoparticles to singlemagnetic nanoparticles.

Example 2 Spin Valve Sensors with Ultrathin Passivation

The reliability of a 4 nm passivation layer has been studied through aseries of passive corrosion studies. A prototype MagArray™ chip wassubmerged into one of two DNA solutions that are currently used instandard DNA microarrays. The first solution, a hybridization buffer(pH=7.5), consists of a mixture of 0.6 M NaCl, 0.06 M C₆H₅Na₃O₇ (sodiumcitrate), and 0.1% SDS (sodium dodecyl sulfate). As its name indicates,this solution is the primary medium for the actual hybridization step inthe microarray. The other solution, a blocking solution (pH=7.9), is aproprietary product from Surmodics (Eden Prairie, Minn.), primarily usedto remove nonspecific binding sites in the test area. This processincreases the likelihood of target molecules interacting with probes.The final addition to these solutions was the DNA (sonicated salmonsperm DNA) at a concentration of 0.1 mg/mL.

The performance of the spin valve sensor after DNA solution exposureswas evaluated by measuring its magnetoresistance (MR) ratio, ΔR/R_(o).This parameter was tracked over time in solution. The first step of theexperiment was to locate an active spin valve sensor (width of about 300nm) with an MR ratio that was reasonably high, about 6-7%. The MR ratiowas measured by use of a probe station setup that gave resistance datawith respect to applied field. The chip was placed in contact with aselected DNA solution for repeated 30 minute cycles after which the chipwas removed, washed with deionized water, air dried, and measured forMR. After 2 hours of cycling, the chip was left in solution for a totalof 24 hours and tested a final time. Note that the sensor currents wereturned off when the detectors were in the solutions becausehybridization and signal detection could be done sequentially.

The testing results for the blocking solution are shown in FIG. 4. Allof the MR values lie between 6-7% with the highest deviation from the0^(th) hour test being around 0.15%. The ΔR values deviate about ±1Ωfrom the 0^(th) hour value. Similarly, the results for the chip inhybridization solution show no dramatic difference from the blockingsolution. The distribution of values is a bit wider for both MR and ΔR.The MR varies no more than 0.4% from the 0^(th) hour and the ΔR scatteris within 4Ω. Thus, the spin valve sensor maintains reasonable levelsfor MR and ΔR through all hours of testing. These results support theMagArray™ design strategy of using ultrathin passivation layer.

Example 3 Synthetic Ferrimagnetic Nanoparticles

Here we disclose novel magnetic nanoparticle tags that are fabricated byphysical methods instead of chemical routes and are suitable forlabeling the target biomolecules to be detected in MagArray™. The tagsconsist of at least two thin ferromagnetic layers, preferablyFe_(x)Co_(1-x), 0.5≦x≦0.7, or Fe_(x)Co_(1-x) based alloys. It is wellknown that Fe_(x)Co_(1-x) has the highest saturation magnetization(about 24.5 kGauss) among the known ferromagnetic materials (Bozorth, R.M., Ferromagnetism, D. Van Nostrand Company, 1951). These ferromagneticlayers are separated by nonmagnetic spacer layers such as Ru, Cr, Au,etc., or their alloys. The spacer layers are appropriately engineered tomake the ferromagnetic layers coupled antiferromagnetically so that thenet remnant magnetization of the resulting particles are zero or nearzero, as shown in FIG. 5. A gold cap is added at the top of theantiferromagnetic stack so that the nanoparticle can be conjugated tobiomolecules via the gold-thiol linkage or other chemical bonding. Theedge of the nanoparticles can be passivated for chemical stability withAu or other thin inert layers. Many physical methods can be conceived bythose familiar with the art to fabricate the nanoparticles describedabove.

Example 4 Additive Fabrication Method of Synthetic FerrimagneticNanoparticles

An additive fabrication method is shown in FIG. 6. As shown therein,going from the top of the figure to the bottom in the direction of thearrows, the fabrication method begins with the deposition of acontinuous thin layer (for releasing particles later) on an ultrasmoothsubstrate, then depositing a mask layer on the release layer. Finally,identical holes are patterned into the mask layer.

In the next step, the film stack is deposited on the mask layer. Thefilm stack, similar to that shown in FIG. 5, includes the ferromagneticlayers, spacer layers, and the gold cap. Following the deposition, themask layer is removed, lifting off the unwanted films deposited on themask layer. Finally, the release layer is removed, lifting off themagnetic nanoparticles into a solution which can then be used asmagnetic tags.

Example 5 Integration of Microfluidic Channels with Detectors

In order to improve the chemical sensitivity of the MagArray™, we haveintegrated microfluidic circuits (Thorsen, T., et al., Science, Vol.298, p. 580 (2002)) directly on the detector array so that DNA probesand target samples are only attached or directed at the detectorsurfaces which are sensitive to magnetic tags, thereby minimizing wasteof DNA probes or DNA targets. The schematic of such systems are shown inFIGS. 7A and 7B. The DNA probes are attached specifically to detectorsurface. In FIG. 7A, the detector is made slightly longer than 20 μmwhich is the width of microfluidic channel. The long detector issuitable for relatively large quantities of DNA samples. In FIG. 7B, thedetector is made shorter than the width of the microfluidic channel. Theshort detector is more suitable for relatively small quantities of DNAsamples. In the latter case, applied electric field or magnetic fieldgradient, and hydrodynamic focusing schemes can be used to direct theDNA samples to the detector surface. It is also conceivable to make themicrofluidic channel in FIG. 7B as wide as the detector length.

All of the compositions and/or methods and/or processes and/or apparatusdisclosed and claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions and methods of this invention have been described in termsof preferred embodiments, it will be apparent to those of skill in theart that variations may be applied to the compositions and/or methodsand/or apparatus and/or processes and in the steps or in the sequence ofsteps of the methods described herein without departing from the conceptand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the scope and concept of the invention.

1. A magnetic tunnel junction detector array, said array comprising: aplurality of detection sites, wherein each detection site of saidplurality comprises: a magnetic tunnel junction detector comprising: abottom electrode; a tunnel barrier layer; and a double-layer topelectrode comprising: a first thin layer, wherein said first thin layeris from about 1 nm to about 10 nm in thickness; and a second layercontacting said first layer, wherein said second layer is a conductivelayer and comprises an aperture exposing a part of said first layer,wherein said tunnel barrier layer is disposed between bottom electrodeand said double-layer top electrode; and a binding molecule covalentlybonded to said magnetic tunnel junction detector.
 2. The magnetic tunneljunction detector array according to claim 1 wherein said tunnel barrierlayer comprises a plurality of magnetic layers contacting the bottomelectrode; a tunnel barrier contacting at least one of the plurality ofmagnetic layers; and a ferromagnetic layer on the top of the tunnelbarrier.
 3. The magnetic tunnel junction detector array according toclaim 1, wherein said first thin layer is a gold layer.
 4. The magnetictunnel junction detector array according to claim 1, wherein saidconductive layer comprises a material selected from the group consistingof aluminum, copper, palladium, platinum, ruthenium, silver, tin,titanium, alloys thereof, oxides thereof, and combinations thereof. 5.The magnetic tunnel junction detector array according to claim 1 whereinsaid binding molecule is covalently bonded to said first thin layerexposed by said aperture.
 6. The magnetic tunnel junction detector arrayaccording to claim 5, wherein said first thin layer is a gold layer. 7.The magnetic tunnel junction detector array according to claim 6 whereinsaid binding molecule is covalently bonded to said gold layer by agold-thiol covalent bond.
 8. The magnetic tunnel junction detector arrayaccording to claim 1 further comprising a row decoder, a column decoder,a preamplifier, and at least one current source.
 9. The magnetic tunneljunction detector array according to claim 1 further comprisingmicrofluidic circuits.